Strand-specific detection of overlapping transcripts via purification involving denaturation of biotinylated cDNA

Abstract

Reverse transcription-PCR (RT-PCR) is the most widely employed technique for gene expression analysis owing to its high sensitivity, easy reproducibility and fast output. It has been conceived that priming RT reactions with gene-specific primers generates cDNA only from the specific RNA. However, several reports have revealed that cDNA is synthesized even without addition of exogenous primers in RT reactions. Owing to such self-priming activity, the signals from specific strands cannot be accurately detected and can confound the expression analysis, especially in context of overlapping bidirectional transcripts. Here, we demonstrate that purification of biotin-tagged cDNA in conjunction with alkaline denaturation can obviate the problem of background priming and enable accurate strand-specific detection of overlapping transcripts.

Keywords

• alkaline denaturation
• biotinylated primers
• detection of overlapping transcripts
• self-priming
• strand-specific reverse transcription-PCR
• streptavidin magnetic beads